Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43–Tyr44 pair and specificity of staphylokinase–plasmin activator complex

Singh, Satish; Ashish, .; Dikshit, Kanak L.

doi:10.1016/j.febslet.2012.01.046

Cited by 10 publications

(26 citation statements)

References 32 publications

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“…Pg activation assay for SAK and SAKVEK was performed in 96‐well microtiter plates as we have described previously [22, 23]. Briefly, SAK or SAKVEK (5 nM) was mixed with Pg (1.5 μM) in a tris assay buffer (50 mM Tris‐HCl, pH 7.5, containing 0.1% BSA, 100 mM NaCl, and 0.01% Tween 80) with 1 mM chromozyme in a microtiter plate, and generation of plasmin was measured by absorbance at 405 nm at 25 °C in a BioTEK Power wave X micro plate reader (VT, USA).…”

Section: Methodsmentioning

confidence: 99%

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Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase

Bhando

Singh

Hade

et al. 2020

Biotech and App Biochem

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Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α‐2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK‐30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C‐terminal end of SAK with a RGD (Arg–Gly–Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK‐30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat/Km, of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2‐ antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin‐specific manner and its ability to clear platelet‐rich plasma clot faster than SAK.

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Section: Methodsmentioning

confidence: 99%

“…Fibrin clot lysis assay was performed as described previously [22]. Briefly, fibrinogen (2 mg/mL) was mixed with 500 nM Pg and 1 mM CaCl 2 in HEPES buffer (pH 7.5) containing 100 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase

Bhando

Singh

Hade

et al. 2020

Biotech and App Biochem

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“…Pg‐activation profile of PadA was generated by mixing 75 n M of PadA with 1.5 µM BPg in HEPES buffer (10 mM HEPES pH 7.5, 0.1% BSA, and 0.01% Tween 80, with or without 100 mM NaCl) containing 1 mM chromozyme in a 96 wells microtiter plate and generation of Pm was measured as a function of time at 405 nm at 25°C in a BioTEK Power wave X microplate reader . To examine the effect of various effectors on Pg activation, the activation profiles of PadA:BPg complex were generated with increasing concentration of Cl − ions, fibrinogen, and CNBr digested fibrinogen.…”

Section: Methodsmentioning

confidence: 99%

Fibrin‐targeted plasminogen activation by plasminogen activator, PadA, from Streptococcus dysgalactiae

2014

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Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three-domain streptokinase (SK) and two-domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to a2-antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single-domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single-domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single-domain plasminogen activator that activates bovine plasminogen in a fibrintargeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin-specific plasminogen activators for thrombolytic therapy.

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“…Only a few studies have been conducted to understand staphylokinase at the molecular level. The results identified that the residues 26, 42–50, 65–69, and 75 are important for the plasmin “partner” binding [[263], [264], [265], [266], [267]], while the residues 11–16, 46–50, 65–69, and 97–98 are involved in the binding and the processing of the plasminogen “substrate” [256,266,[268], [269], [270]]. In contrast, the first ten N-terminal residues are not required for the activity and are cleaved by active plasmin [268,[271], [272], [273], [274], [275]].…”

Section: Staphylokinasementioning

confidence: 99%

Structural Biology and Protein Engineering of Thrombolytics

Mičan

Toul

Bednář

et al. 2019

Computational and Structural Biotechnology Journal

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Myocardial infarction and ischemic stroke are the most frequent causes of death or disability worldwide. Due to their ability to dissolve blood clots, the thrombolytics are frequently used for their treatment. Improving the effectiveness of thrombolytics for clinical uses is of great interest. The knowledge of the multiple roles of the endogenous thrombolytics and the fibrinolytic system grows continuously. The effects of thrombolytics on the alteration of the nervous system and the regulation of the cell migration offer promising novel uses for treating neurodegenerative disorders or targeting cancer metastasis. However, secondary activities of thrombolytics may lead to life-threatening side-effects such as intracranial bleeding and neurotoxicity. Here we provide a structural biology perspective on various thrombolytic enzymes and their key properties: (i) effectiveness of clot lysis, (ii) affinity and specificity towards fibrin, (iii) biological half-life, (iv) mechanisms of activation/inhibition, and (v) risks of side effects. This information needs to be carefully considered while establishing protein engineering strategies aiming at the development of novel thrombolytics. Current trends and perspectives are discussed, including the screening for novel enzymes and small molecules, the enhancement of fibrin specificity by protein engineering, the suppression of interactions with native receptors, liposomal encapsulation and targeted release, the application of adjuvants, and the development of improved production systems.

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Pro⁴² and Val⁴⁵ of staphylokinase modulate intermolecular interactions of His⁴³–Tyr⁴⁴ pair and specificity of staphylokinase–plasmin activator complex

Cited by 10 publications

References 32 publications

Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase

Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase

Fibrin‐targeted plasminogen activation by plasminogen activator, PadA, from Streptococcus dysgalactiae

Structural Biology and Protein Engineering of Thrombolytics

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